1. Field of the Invention
This invention relates to a method for assaying an enzyme in a sample and a kit for use therein. More particularly, this invention relates to a kinetic method for assaying prostate acid phosphatase (PAP) in a sample and to a kit for use therein.
2. Description of the Prior Art
Methods for the assay of acid phosphatase and prostatic acid phosphatase (PAP) have been reported (1-5). Choe et al. (1) disclose an immunoenzyme assay for human PAP wherein anti-PAP.about.PAP complexes are precipitated with a 100% saturated solution of (NH.sub.4).sub.2 SO.sub.4. After removing a supernatant, pellets are dissolved in acetate or citrate buffer, pH 5.0. The enzyme assay is initiated by the addition of p-nitrophenylphosphate and the enzyme reaction is stopped by the addition of sodium hydroxide.
Choe et al. (2) and Ly et al. (3) disclose an immuno-assisted assay (IAA) for PAP basically consisting of the following reaction sequence: ##STR1## wherein GAR denotes goat anti-rabbit Ig and Pi denotes inorganic phosphate.
The above immunoassays suffer from various pitfalls, including, the requirement of a sample blank; a time-consuming procedure; and the requirement of a potentially hazardous alkaline solution (NaOH) to stop the reaction and color development of the chromophore (p-nitrophenol).
Sanders et al. (4) and Quan et al. (5) disclose a kinetic determination method for acid phosphatase with .alpha.-naphthyl phosphate as substrate and a diazonium salt for color development. Quan et al. also disclose a method for indirectly calculating PAP acitivity. In addition, Quan et al. also disclose that better sensitivity and specificity can be achieved by activating the PAP and suppressing the non-prostatic acid phosphatase with 1,5-pentanediol, and that better solubility of the azo dye complex is achieved by the addition of a detergent, namely Brij-35.
The methods of Sanders et al. and Quan et al. suffer from various pitfalls including the instability of the complete reagent for the kinetic acid phosphatase determination which necessitates the use of the reagent blank, the interaction of plasma and serum components and diazonium salts which makes advisable the use of a sample blank measured in the absence of substrate for every determination, and the inability to directly measure PAP.
It would be advantageous to have a kinetic assay for the determination of PAP which eliminates most, if not all, of the disadvantages present in the above-described methodology.